Angiogenesis imaging holds considerable promise for early detection of cancer, as well as post-therapy assessment of many new molecular-targeted antiangiogenic therapies. New contrast probes such as small molecular radiotracers, optical probes, and lipid- and polymer-based nanoparticles are intensively investigated to target different biomarkers of angiogenesis. However, low tissue concentrations of intended biomarkers, lack of an amplification strategy to increase signal output, and high background signals are several major limiting factors that hamper the advances of these molecular imaging techniques. The long-term goal of this application is to develop a robust set of tunable fluorescent nanoprobes based on the homo fluorescent resonance energy transfer (homoFRET) and photo-induced electron transfer (PET) mechanisms. The micelle nanoprobes will stay silent (or in the OFF state) with minimum background signals under normal physiological conditions (e.g. blood circulation). Upon specific targeting to angiogenic target (e.g. avb3), these nanoprobes can be turned ON by pH activation (pH 5.0-7.2) inside endosomes/lysosomes after receptor-mediated endocytosis. Our central hypothesis is that a synergized strategy of signal amplification in tumor endothelium and background suppression in blood and pH-activatable micelle (pHAM) nanoprobes will be able to improve the imaging sensitivity and specificity of angiogenesis biomarkers in vascularized tumors in vivo. To test this hypothesis, we will carry out the following specific aims: (1) establish a series of near infrared (NIR) pHAM nanoprobes with tunable transition pH (pHt);(2) evaluate the activation of non-targeted pHAM in acidic tumor microenvironment;(3) establish vascular-targeted pHAM and investigate the intracellular activation of these nanoprobes in tumor endothelial cells;(4) evaluate the specificity and efficacy of targeted pHAM in the imaging of distinctive angiogenesis biomarkers (i.e. VEGFR2, avb3) in tumor-bearing mice in vivo. Successful execution of this research will establish pHAM as a valuable imaging platform to image angiogenesis- specific biomarkers on the tumor endothelium in vivo. These nanoprobes may be particularly useful for the efficacy assessment of molecular-targeted antiangiogenic therapies, where the expression levels of the therapeutic targets (e.g. VEGFR2, avb3) can be directly measured. PUBLIC HEALTH RELEVANCE: This application describes the development of ultra-responsive fluorescent nanoparticles for molecular imaging of angiogenesis biomarkers in preclinical tumor models.